Medical implants containing adenosine receptor agonists and methods for inhibitiing medical implant loosening

ABSTRACT

The invention provides methods and compositions for reducing or inhibiting bone resorption, osteoclast differentiation and stimulation and the loosening of medical prostheses by administering a compound or agent that modulates an adenosine receptor such as the adenosine A 2A  receptor, in particular, an agonist of an adenosine A 2A  receptor. The invention also extends to pharmaceutical compositions comprising such an agent that modulates an adenosine receptor such as an adenosine A 2A  agonist and to prosthetic devices containing such an agent that modulates an adenosine receptor such as an A 2A  agonist on one or more surfaces or within the prosthetic device such as, for example, suspended in the cement forming the prosthetic device.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for reducingor inhibiting bone resorption, osteoclast differentiation andstimulation and the loosening of medical prostheses.

BACKGROUND OF THE INVENTION

Adenosine is a nucleoside that occurs naturally in mammals, which actsas a ubiquitous biochemical messenger. The heart, for instance, producesand releases adenosine in order to modulate heart rate and coronaryvasodilation. Likewise, adenosine is produced in the kidney to modulateessential physiological responses, including glomerular filtration rate(GFR), electrolyte reabsorption, and renin secretion.

Adenosine is known to bind to and activate seven-transmembrane spanningG-protein coupled receptors, thereby eliciting a variety ofphysiological responses. There are 4 known subtypes of adenosinereceptors (i.e., A₁, A_(2A), A_(2B), and A₃), which mediate different,and sometimes opposing, effects. For example, activation of theadenosine A₁ receptor, elicits an increase in renal vascular resistance,which leads to a decrease in glomerular filtration rate (GFR), whileactivation of the adenosine A_(2A) receptor elicits a decrease in renalvascular resistance. Conversely, blockade of the A₁ adenosine receptordecreases afferent arteriole pressure, leading to an increase in GFR andurine flow, and sodium excretion. Furthermore, A_(2A) adenosinereceptors modulate coronary vasodilation, whereas A_(2B) receptors havebeen implicated in mast cell activation, asthma, vasodilation,regulation of cell growth, intestinal function, and modulation ofneurosecretion (See, Adenosine A_(2B) Receptors as Therapeutic Targets,Drug Dev Res 45:198; Feoktistov et al., Trends Pharmacol Sci 19:148-153and Ralevic, V. and Burnstock, G. (1998), Pharmacological Reviews, Vol.50: 413-492), and A₃ adenosine receptors modulate cell proliferationprocesses. Two receptor subtypes (A₁ and A_(2A)) exhibit affinity foradenosine in the nanomolar range while two other known subtypes A_(2B)and A₃ are low-affinity receptors, with affinity for adenosine in thelow-micromolar range. A₁ and A₃ adenosine receptor activation can leadto an inhibition of adenylate cyclase activity, while A_(2A) and A_(2B)activation causes a stimulation of adenylate cyclase.

It has been shown that adenosine, acting at specific cell surfacereceptors, has the potential to suppress inflammation and thatinflammation itself may increase extracellular adenosine levels(Cronstein, et al., 1986, Journal of Clinical Investigation 78:760-770;Cronstein, et al., 1983, Journal of Experimental Medicine158:1160-1177). Further, it has been demonstrated that adenosinemediates the anti-inflammatory effects of low-dose methotrexate therapyfor Rheumatoid Arthritis (Reviewed in Cronstein, 2005, Pharmacol Rev57:163-172). Exploration of the therapeutic and toxic properties ofmethotrexate in the treatment of RA has led to a number of otherpotentially important pre-clinical therapeutic developments.Methotrexate increases giant cell formation from peripheral bloodmonocytes and that this effect is mediated by adenosine acting at A₁receptors (Merrill, et al., Arth. Rheum. 40:1308-1315). In addition,A_(2A) receptor antagonists promote giant cell formation by diminishingthe effect of endogenous adenosine although the A₁ receptor-mediatedpromotion of giant cell formation appears to dominate.

A₁ receptor antagonists completely block, in a dose-dependent fashion,osteoclast formation. Similarly, the A₁ receptor antagonists blockosteoclast function (resorption of dentin). Six-month old A₁ KO micedemonstrate increased bone density. Their bones demonstrate diminishedresorption, and some evidence indicates that the osteoclasts in the A₁knockout mice do not resorb bone. A murine model of post-menopausalosteoporosis, ovariectomy-induced bone loss, reveals that treatment ofmice with an adenosine A₁ receptor antagonist completely preventsovariectomy-induced bone loss. Adenosine A₁ receptors may be useful intreating and preventing osteoporosis.

Replacement of osteoarthritic or damaged hips and knees is among themost common surgical procedures performed in the United States and otherdeveloped countries. Excellent results are achieved in more than 95% ofpatients. Prosthodontic prostheses are also increasingly successful aswell. However, aside from infectionm the most pressing difficulty inmaintaining mobility is the development of bone resorption and looseningof the prosthesis leading to early reimplantation. There are twocomponents to the bone resorption including an inflammatory reaction todebris from the prosthesis (ultra high molecular weight polyethylenedebris). However, inflammation alone is not sufficient to induce boneresorption. Differentiation and stimulation of osteoclasts is requiredfor the destruction of peri-prosthetic bone and bone loosening.

All publications, patent applications, patents and other referencematerial mentioned are incorporated by reference in their entirety. Inaddition, the materials, methods and examples are only illustrative andare not intended to be limiting. The citation of references herein shallnot be construed as an admission that such is prior art to the presentinvention.

SUMMARY OF THE INVENTION

The invention relates to the application and use of modulators of anadenosine receptor, including agonists of an adenosine receptor, toinhibit bone resorption, inhibit differentiation and stimulation ofosteoclasts, and to reduce loosening of medical prostheses.

In a first aspect, the invention provides a method for inhibiting boneresorption comprising administering to a subject a therapeuticallyeffective amount of an adenosine receptor agonist, or an analog,derivative or combination thereof.

In a second aspect, the invention provides a method for inhibitingdifferentiation and stimulation of osteoclasts comprising administeringto the subject a therapeutically effective amount of an adenosinereceptor agonist, or an analog, derivative or combination thereof.

In a third aspect, the invention provides a method of inhibiting orreducing loosening of a medical prosthesis comprising administering to asubject an amount of an adenosine receptor agonist effective to inhibitor reduce bone resorption, or an analog, derivative or combinationthereof.

For each of these three aspects, in one particular embodiment, theadenosine receptor of the present invention is selected from the groupconsisting of A₁, A_(2A), A_(2B) and A₃. In a more particularembodiment, the adenosine receptor is an A_(2A) receptor, and theagonist is an adenosine receptor A_(2A) agonist. In yet anotherembodiment, the adenosine receptor agonist inhibits more than oneadenosine receptor.

In another particular embodiment, the adenosine receptor agonist is aselective adenosine receptor agonist. In still other particularembodiments, the adenosine receptor agonist is a non-selective adenosinereceptor agonist.

In a more particular embodiment, the agent that agonizes an adenosinereceptor is an adenosine A_(2A) receptor agonist. The adenosine receptoragonist may be, for instance, a small organic molecule, a protein orpeptide, a nucleic acid or an antibody.

In yet another more particular embodiment, the adenosine receptoragonist is capable of substantially stimulating the endogenous activityof the adenosine receptor substantially the same as though the adenosinereceptor had encountered its natural, endogenous ligand.

In yet another particular embodiment, the adenosine receptor agonist, inmany embodiments an adenosine A_(2A) receptor agonist is administeredvia an implanted device.

In one particular embodiment, an effective amount of an adenosinereceptor agonist may be used in combination with one or more drugsuseful in inhibiting bone resorption or inhibiting differentiation orstimulation of osteoclasts or a combination of any of these agents.

Adenosine A_(2A) receptor agonists are well known in the art. Many aredisclosed in, for instance, U.S. Pat. Nos. 7,226,913 and 6,326,359 andin United States Patent Publication Nos. 20070225247, 20060100169,20060034941, 20050261236, 20050182018, 20050171050, 20050020915 and20040064039, the disclosures of which are herein incorporated byreference in their entireties. In another more particular embodiment,the drug is selected from the group consisting of CGS 21680, MRE-0094,IB-MECA and R-PIA, binodenoson, ATL146, for instance.

The adenosine receptor agonist may be administered alone or incombination with one or more other compounds or agents for inhibitingbone resorption, osteoclast differentiation and stimulation andprosthesis loosening. Such other compounds may be, for instance,anti-inflammatory compounds, bisphosphonates or growth factors. Theadenosine receptor agonist may be administered with a second adenosinereceptor agonist or with a less selective adenosine receptor agonist.(i.e. one that binds other adenosine receptors in addition to A_(2A),for example A_(2B), A₁ or A₃).

In one embodiment, the adenosine receptor agonist may be selective forthe receptor, or it may be a non-selective adenosine receptor agonist,which may stimulate or mimic natural ligands of one or more of thefollowing receptors: A₁, A_(2A), A_(2B) or A₃. In a preferredembodiment, the adenosine receptor agonist is an adenosine A_(2A)receptor agonist.

A fourth aspect of the invention provides a cement, absorbable matrix orother substance containing an adenosine receptor modulator, inparticular, an adenosine receptor agonist. In many instances, thecement, absorbable matrix or other substance containing an adenosinereceptor modulator is present in a prosthetic device. The prostheticdevice is useful for inhibiting osteoclast differentiation andstimulation, bone resorption, and prosthesis loosening. The adenosinereceptor modulator such as an adenosine A_(2A) receptor agonist may bepresent in a composition applied to one or more surfaces of theprosthetic device or the adenosine receptor modulating agent such as anadenosine A_(2A) receptor agonist may be present within the cement ormatrix. In some embodiments, the adenosine receptor modulating agentsuch as an adenosine A_(2A) receptor agonist, may be present within thevery matrix of the prosthetic device such as for instance within thecement, e.g. methylmethacrylate cement that forms the prosthetic device.

In a fifth aspect, the present invention provides a pharmaceuticalcomposition comprising the adenosine receptor agonist alone or incombination with one or more compounds or agents effective forinhibiting bone resorption, osteoclast differentiation and stimulationand the loosening of medical prostheses. The adenosine receptor agonistand the one or more compounds or agents may be formulated andadministered alone or together. The pharmaceutical composition(s)comprising the adenosine receptor agonist and the one or more compoundsor agents may be administered concurrently or sequentially. In anotherparticular embodiment, the one or more compounds or agents effective forinhibiting bone resorption, osteoclast differentiation and stimulationand the loosening of medical prostheses are selected from the groupconsisting of those effective for stimulating bone density and thoseeffective for inhibiting or reducing inflammation. The pharmaceuticalcompositions may be delivered orally or parenterally. They may bedelivered via the intravenous route, the intramuscular route, or thesubcutaneous route. They may be delivered as an immediate releaseformulation or as a slow or sustained release formulation. In someparticular embodiments, the compositions are delivered on the surface ofa prosthetic device or are delivered in the very matrix of a prostheticdevice.

In another more particular embodiment, the pharmaceutical compositioncomprising the adenosine receptor agonist may also contain one or moredrugs selected from the group consisting of anti-inflammatory agents,growth factors, bone morphogenetic protein, soluble RANK.

Other objects and advantages will become apparent to those skilled inthe art from a review of the following description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. FIG. 1A provides measured mRNA levels of all four adenosinereceptors obtained from osteoclasts derived from bone marrow osteoclastprecursors, splenocyte osteoclast precursors or the murine cell lineRAW264.7 which differentiates into osteoclast like cells therebydemonstrating expression of adenosine receptors in each cell line. FIG.1B provides Western Blot analysis of bone marrow-derived osteoclastlysates demonstrating the presence of all four adenosine receptors.

FIG. 2 demonstrates that both bone mineral density (FIG. 2A) and bonemineral Ca⁺⁺ (FIG. 2B) are diminished in adenosine A_(2A) receptorknockout mice suggesting that adenosine receptor knockout mice eitherhave increased bone absorption or diminished bone production.

FIG. 3 demonstrates that adenosine A_(2A) receptor knockout mice haveevidence for increased bone resorption and increased numbers ofosteoclasts in their bones. Alcian Blue staining of decalcified bonefrom WT (FIG. 3 A, B) and A_(2A) knockout (FIG. 3 C, D) micedemonstrates a marked increase in the number of osteoclasts anddiminished bone substance in the A_(2A) receptor knockout mice.

FIG. 4 demonstrates the increased number of osteoclasts correlates withdiminished bone mineral density. The increased number of osteoclastspresent in the bones of A_(2A) KO mice, as compared to WT mice (FIG. 4A)correlates with the diminished bone mineral density observed by DEXAscan of these mice (FIG. 4B).

FIG. 5 provides electron microscopy demonstrating greater apparentactivity of osteoclasts in A_(2A) KO mice. Examination of bone byelectron microscopy demonstrates larger bone resorption pits (clearareas) in the bones of A_(2A) receptor knockout mice (FIG. 5B) than wildtype mice (FIG. 5A).

FIG. 6 demonstrates that adenosine A_(2A) receptor blockade diminishesosteoclast formation in vitro by RAW264.7 cells induced to undergoosteoclast differentiation. Adenosine A_(2A) receptor antagonist treatedRAW264.7 cells (FIG. 6B) form an increased number of multinucleatedTRAP+ osteoclasts in culture than untreated RAW264.7 cells (FIG. 6A).This is graphically demonstrated in FIG. 6C.

FIG. 7 demonstrates that adenosine A_(2A) receptor blockadedownregulates TRAF6 protein in RAW264.7 cells (FIG. 7A) as compared toβ-actin (FIG. 7B). A₁ adenosine receptor blockade diminishes TRAF6protein, a critical signaling protein for osteoclast differentiation, inbone marrow osteoclasts and RAW264.7 cells. To determine whetheradenosine A_(1A) receptors regulate osteoclast differentiation by asimilar mechanism, TRAF6 levels in RAW264.7 cells treated with anadenosine A₁ receptor antagonist, an A_(2A) agonist and antagonist weredetermined. Both the A₁ receptor antagonist and A_(2A) receptor agonistdiminish TRAF6 levels in RAW264.7 cells.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods and treatment methodology are described, itis to be understood that this invention is not limited to particularmethods and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused herein is for purposes of describing particular embodiments only,and is not intended to be limiting, since the scope of the presentinvention will be limited only by the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein and/or which will become apparent to those persons skilled in theart upon reading this disclosure and so forth in their entirety.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference I their entireties.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

DEFINITIONS

The terms used herein have the meanings recognized and known to those ofskill in the art, however, for convenience and completeness, particularterms and their meanings are set forth below.

“Agent” refers to all materials that may be used to preparepharmaceutical and diagnostic compositions, or that may be compoundssuch as small synthetic or naturally derived organic compounds, nucleicacids, polypeptides, antibodies, fragments, isoforms, variants, or othermaterials that may be used independently for such purposes, all inaccordance with the present invention.

By “agonist” is meant a substance that binds to a specific receptor andtriggers a response in a cell. It mimics the action of an endogenousligand (such as hormone or neurotransmitter) that binds to the samereceptor. A “full agonist” binds (has affinity for) and activates areceptor, displaying full efficacy at that receptor. One example of adrug that acts as a full agonist is isoproterenol which mimics theaction of acetylcholine at β adrenoreceptors. A “partial agonist” (suchas buspirone, aripiprazole, buprenorphine, or norclozapine) also bindsand activates a given receptor, but has only partial efficacy at thereceptor relative to a all agonist. A “partial agonist” may also beconsidered a ligand that displays both agonistic and antagonisticeffects—when both a full agonist and partial agonist are present, thepartial agonist actually acts as a competitive antagonist, competingwith the full agonist for receptor occupancy and producing a netdecrease in the receptor activation observed with the full agonistalone. A “co-agonist” works with other co-agonists to produce thedesired effect together. An antagonist blocks a receptor from activationby agonists. Receptors can be activated or inactivated either byendogenous (such as hormones and neurotransmitters) or exogenous (suchas drugs) agonists and antagonists, resulting in stimulating orinhibiting a biological response. A ligand can concurrently behave asagonist and antagonist at the same receptor, depending on effectorpathways.

The potency of an agonist is usually defined by its EC₅₀ value. This canbe calculated for a given agonist by determining the concentration ofagonist needed to elicit half of the maximum biological response of theagonist. Elucidating an EC₅₀ value is useful for comparing the potencyof drugs with similar efficacies producing physiologically similareffects. The lower the EC₅₀, the greater the potency of the agonist andthe lower the concentration of drug that is required to elicit a maximumbiological response.

“Antagonist” refers to an agent that down-regulates (e.g., suppresses orinhibits) at least one bioactivity of a protein. An “antagonist” or anagent that “antagonizes” may be a compound which inhibits or decreasesthe interaction between a protein and another molecule, e.g., a targetpeptide or enzyme substrate. An antagonist may also be a compound thatdown-regulates expression of a gene or which reduces the amount ofexpressed protein present. Methods for assessing the ability of an agentto “antagonize” or “inhibit” an adenosine receptor are known to thoseskilled in the art.

“Analog” as used herein, refers to a chemical compound, a nucleotide, aprotein, or a polypeptide that possesses similar or identical activityor function(s) as the chemical compounds, nucleotides, proteins orpolypeptides having the desired activity and therapeutic effect of thepresent invention (e.g. to treat or prevent bone disease, or to modulateosteoclast differentiation), but need not necessarily comprise acompound that is similar or identical to those compounds of thepreferred embodiment, or possess a structure that is similar oridentical to the agents of the present invention.

“Derivative” refers to the chemical modification of molecules, eithersynthetic organic molecules or proteins, nucleic acids, or any class ofsmall molecules such as fatty acids, or other small molecules that areprepared either synthetically or isolated from a natural source, such asa plant, that retain at least one function of the active parentmolecule, but may be structurally different. Chemical modifications mayinclude, for example, replacement of hydrogen by an alkyl, acyl, oramino group. It may also refer to chemically similar compounds whichhave been chemically altered to increase bioavailability, absorption, orto decrease toxicity. A derivative polypeptide is one modified byglycosylation, pegylation, or any similar process that retains at leastone biological or immunological function of the polypeptide from whichit was derived.

By “medical prosthetic device” or “prosthesis” is meant an artificialcomponent, device or extension that replaces a portion or all of a bodypart whether the body part is entirely or partially missing. The termincludes artificial limbs, breast prosthesis such as those implantedpost-mastectomy, cochlear implants, corrective lenses, craniofacialprosthesis, dental/maxillofacial prosthetics such as those implanted tocorrect a cleft palate, dentures, dental restoration, facialprosthetics, hair prosthesis, neuroprosthetics, ocular prosthetics,ostomies such as colostomy, ileostomy and urostomy, penile prosthetics,replacement joints such as hips, knees and shoulders, simatoprosthetics, prosthetic testis and transtibial prosthesis.

A “small molecule” refers to a molecule that has a molecular weight ofless than 3 kilodaltons (kDa), preferably less than about 1.5kilodaltons, more preferably less than about 1 kilodalton. Smallmolecules may be nucleic acids, peptides, polypeptides, peptidomimetics,carbohydrates, lipids or other organic (carbon-containing) or inorganicmolecules. As those skilled in the art will appreciate, based on thepresent description, extensive libraries of chemical and/or biologicalmixtures, often fungal, bacterial, or algal extracts, may be screenedwith any of the assays of the invention to identify compounds thatmodulate a bioactivity. A “small organic molecule” is normally anorganic compound (or organic compound complexed with an inorganiccompound (e.g., metal)) that has a molecular weight of less than 3kilodaltons, and preferably less than 1.5 kilodaltons, and morepreferably less than about 1 kDa.

“Diagnosis” or “screening” refers to diagnosis, prognosis, monitoring,characterizing, selecting patients, including participants in clinicaltrials, and identifying patients at risk for or having a particulardisorder or clinical event or those most likely to respond to aparticular therapeutic treatment, or for assessing or monitoring apatient's response to a particular therapeutic treatment.

The concept of “combination therapy” is well exploited in currentmedical practice. Treatment of a pathology by combining two or moreagents that target the same pathogen or biochemical pathway sometimesresults in greater efficacy and diminished side effects relative to theuse of the therapeutically relevant dose of each agent alone. In somecases, the efficacy of the drug combination is additive (the efficacy ofthe combination is approximately equal to the sum of the effects of eachdrug alone), but in other cases the effect can be synergistic (theefficacy of the combination is greater than the sum of the effects ofeach drug given alone). As used herein, the term “combination therapy”means the two compounds can be delivered in a simultaneous manner, e.g.concurrently, or one of the compounds may be administered first,followed by the second agent, e.g sequentially. The desired result canbe either a subjective relief of one or more symptoms or an objectivelyidentifiable improvement in the recipient of the dosage.

“Differentiate” or “differentiation” as used herein, generally refers tothe process by which precursor or progenitor cells differentiate intospecific cell types. In the matter of the present invention, the termrefers to the process by which pre-osteoclasts become osteoclasts.Differentiated cells can be identified by their patterns of geneexpression and cell surface protein expression. As used herein, the term“differentiate” refers to having a different character or function fromthe original type of tissues or cells. Thus, “differentiation” is theprocess or act of differentiating. The term “Osteoclast Differentiation”refers to the process whereby osteoclast precursors in the bone marrowbecome functional osteoclasts.

“Modulation” or “modulates” or “modulating” refers to up regulation(i.e., activation or stimulation), down regulation (i.e., inhibition orsuppression) of a response, or the two in combination or apart. As usedherein, an adenosine receptor “modulator” or “modulating” compound oragent is a compound or agent that modulates at least one biologicalmarker or biological activity characteristic of osteoclasts and boneformation. The term “modulating” as related to osteoclastdifferentiation, refers to the ability of a compound or agent to exertan effect on precursors to osteoclasts, or to alter the expression of atleast one gene related to osteoclastogenesis. For example, expression ofthe following genes is modulated during osteoclastogenesis: DC-Stamp,tartrate resistant alkaline phosphatase (TRAP), cathepsin K, calcitoninreceptor, and integrin.

As used herein, the term “candidate compound” or “test compound” or“agent” or “test agent” refers to any compound or molecule that is to betested. As used herein, the terms, which are used interchangeably, referto biological or chemical compounds such as simple or complex organic orinorganic molecules, peptides, proteins, oligonucleotides,polynucleotides, carbohydrates, or lipoproteins. A vast array ofcompounds can be synthesized, for example oligomers, such asoligopeptides and oligonucleotides, and synthetic organic compoundsbased on various core structures, and these are also included in theterms noted above. In addition, various natural sources can providecompounds for screening, such as plant or animal extracts, and the like.Compounds can be tested singly or in combination with one another.Agents or candidate compounds can be randomly selected or rationallyselected or designed. As used herein, an agent or candidate compound issaid to be “randomly selected” when the agent is chosen randomly withoutconsidering the specific interaction between the agent and the targetcompound or site. As used herein, an agent is said to be “rationallyselected or designed”, when the agent is chosen on a nonrandom basiswhich takes into account the specific interaction between the agent andthe target site and/or the conformation in connection with the agent'saction.

“Treatment” or “treating” refers to therapy, prevention and prophylaxisand particularly refers to administering medicine or performing medicalprocedures on a patient, for either prophylaxis (prevention) or to cureor reduce the extent of or likelihood of occurrence of the infirmity ormalady or condition or event. In the present invention, the treatmentsusing the agents described may be provided to slow or halt bone loss, orto increase the amount or quality of bone density. Most preferably, thetreating is for the purpose of reducing or diminishing bone resorptionand resultant prosthetic device loosening. Treating as used herein alsomeans administering the compounds for increasing bone density or formodulating osteoclastogenesis in individuals. Furthermore, in treating asubject, the compounds of the invention may be administered to a subjectalready suffering from loss of bone mass or other bone disease asprovided herein or to prevent or inhibit the occurrence of suchcondition.

“Subject” or “patient” refers to a mammal, preferably a human, in needof treatment for a condition, disorder or disease.

“Osteoclastogenesis” refers to osteoclast generation, which is amulti-step process that can be reproduced in vitro. Earlier in vitroosteoclastogenesis systems used mixtures of stromal or osteoblasticcells together with osteoclast precursors from bone marrow (Suda, etal., (1997) Methods Enzymol. 282, 223-235; David et al., (1998) J. BoneMiner. Res. 13, 1730-1738). These systems utilized 1α,25-dihydroxyvitamin D₃ to stimulate stromal/osteoblastic cells toproduce factors that support osteoclast formation More recent modelsutilize bone marrow cells cultured with soluble forms of the cytokinesM-CSF (macrophage-colony stimulating factor) and a soluble form of RANKL(receptor activator of nuclear factor KB ligand) (Lacey, et al., (1998)Cell 93, 165-176; Shevde et al., (2000) Proc. Natl. Acad. Sci. U.S.A.97, 7829-7834). These two cytokines are now recognized as the majorfactors from stromal cells that support osteoclastogenesis (Takahashi,et al., (1999) Biochem. Biophys. Res. Commun. 256, 449-455). Thus, theiraddition to the culture medium overcomes the need for stromal cells.

“Osteoclast precursor” refers to a cell or cell structure, such as apre-osteoclast, which is any cellular entity on the pathway ofdifferentiation between a macrophage and a differentiated and functionalosteoclast. The term osteoclast includes any osteoclast-like cell orcell structure which has differentiated fully or partially from amacrophage, and which has osteoclast character, including but notlimited to positive staining for tartrate-resistant acid phosphatase(TRAP), but which is not a fully differentiated or functionalosteoclast, including particularly aberrantly differentiated or nonfunctional osteoclasts or pre-osteoclasts.

“Osteoclast culture” refers to any in vitro or ex vivo culture or systemfor the growth, differentiation and/or functional assessment ofosteoclasts or osteoclast precursors, whether in the absence or presenceof other cells or cell types, for instance, but not limited to,osteoblasts, macrophages, hematopoietic or stromal cells.

“Osteoclast function”, as used herein, refers to bone resorption and theprocesses required for bone resorption.

An “amount sufficient to inhibit osteoclast differentiation, formationor function” refers to the amount of the adenosine receptor agonistsufficient to block either the differentiation, the formation or thefunction of osteoclasts, more particularly, an amount ranging from about0.1 nM to about 10 μM, or more preferentially from about 0.1 nM to about5 μM, and most preferentially from about 0.1 nM to about 1 μM in vitro.In vivo amounts of an adenosine receptor agonist such as an adenosineA_(2A) receptor agonist sufficient to block either the differentiation,the formation or the function of osteoclasts may range from about 0.1mg/Kg of body weight per day to about 200 mg/Kg of body weight per dayin vivo, or more preferentially from about 1 mg/Kg to about 100 mg/Kg,and most preferentially from about 25 mg/Kg to about 50 mg/Kg of bodyweight per day in vivo. It is understood that the dose, whenadministered in vivo, may vary depending on the clinical circumstances,such as route of administration, age, weight and clinical status of thesubject in which inhibition of osteoclast differentiation, formation orfunction is desired.

In a specific embodiment, the term “about” means within 20%, preferablywithin 10%, and more preferably within 5%.

An “effective amount” or a “therapeutically effective amount” is anamount sufficient to decrease or prevent the symptoms associated withthe conditions disclosed herein, including bone loss or in a decrease inbone mass or density, such as that which occurs with medical prostheticdevices or other related conditions contemplated for therapy with thecompositions of the present invention. For example, an “effectiveamount” for therapeutic uses is the amount of the composition comprisingan active compound herein required to provide reversal or inhibition ofbone loss or delay the onset of prosthetic device loosening, increaseand/or accelerate bone growth into prosthetic devices, etc. Sucheffective amounts may be determined using routine optimizationtechniques and are dependent on the particular condition to be treated,the condition of the subject, the route of administration, theformulation, and the judgment of the practitioner and other factorsevident to those skilled in the art. The dosage required for thecompounds of the invention is that which induces a statisticallysignificant difference in bone mass or inhibition of bone loss betweentreatment and control groups. This difference in bone mass or bone lossmay be seen, for example, as at least 1-2%, or any clinicallysignificant increase in bone mass or reduction in bone loss in thetreatment group. Other measurements of clinically significant increasesin healing may include, for example, an assay for the N-terminalpropeptide of Type I collagen, tests for breaking strength and tension,breaking strength and torsion, 4-point bending, increased connectivityin bone biopsies and other biomechanical tests well known to thoseskilled in the art. General guidance for treatment regimens may beobtained from experiments carried out in vitro or in animal models ofthe disease of interest. The “effective amount” or “therapeuticallyeffective amount” may range from about 1 mg/Kg to about 200 mg/Kg invivo, or more preferentially from about 10 mg/Kg to about 100 mg/Kg, andmost preferentially from about 25 mg/Kg to about 50 mg/Kg in vivo.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water or aqueous solution saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

Binding compounds can also be characterized by their effect on theactivity of the target molecule. Thus, a “low activity” compound has aninhibitory concentration (IC₅₀) (for inhibitors or antagonists) oreffective concentration (EC₅₀) (applicable to agonists) of greater than1 μM under standard conditions. By “very low activity” is meant an IC₅₀or EC₅₀ of above 100 μM under standard conditions. By “extremely lowactivity” is meant an IC₅₀ or EC₅₀ of above 1 mM under standardconditions. By “moderate activity” is meant an IC₅₀ or EC₅₀ of 200 nM to1 μM under standard conditions. By “moderately high activity” is meantan IC₅₀ or EC₅₀ of 1 nM to 200 nM. By “high activity” is meant an IC₅₀or EC₅₀ of below 1 nM under standard conditions. The IC₅₀ (or EC₅₀) isdefined as the concentration of compound at which 50% of the activity ofthe target molecule (e.g., enzyme or other protein) activity beingmeasured is lost (or gained) relative to activity when no compound ispresent. Activity can be measured using methods known to those ofordinary skill in the art, e.g., by measuring any detectable product orsignal produced by occurrence of an enzymatic reaction, or otheractivity by a protein being measured.

An individual “at risk” may or may not have detectable disease, and mayor may not have displayed detectable disease prior to the treatmentmethods described herein. “At risk” denotes that an individual who isdetermined to be more likely to develop a symptom based on conventionalrisk assessment methods or has one or more risk factors that correlatewith development of a bone disease or low bone mass or density orenhanced susceptibility to bone resorption. An individual having one ormore of these risk factors has a higher probability of developing boneresporption than an individual without these risk factors.

“Prophylactic” or “therapeutic” treatment refers to administration tothe host of one or more of the subject compositions. If it isadministered prior to clinical manifestation of the unwanted condition(e.g., disease or other unwanted state of the host animal) then thetreatment is prophylactic, i.e., it protects the host against developingthe unwanted condition, whereas if administered after manifestation ofthe unwanted condition, the treatment is therapeutic (i.e., it isintended to diminish, ameliorate or maintain the existing unwantedcondition or side effects therefrom).

General Description

The invention relates to the unexpected finding that stimulating anadenosine receptor such as an adenosine A_(2A) receptor with agents thatare agonists of the receptor leads to or results in inhibition ofosteoclast differentiation, formation, or function, leads to less boneresorption, and subsequently leads to less loosening of surgicallyimplanted medical prostheses. As such, these agonists may be used totreat a subject having a condition characterized by bone loss that maylead to subsequent loosening of a medical prosthesis. These agonists maybe especially well suited for treating bone loss associated withprosthetic implantation, other forms of osteopenia, and in otherconditions where facilitation of bone repair or replacement is desiredsuch as bone fractures, bone defects, plastic surgery, dental and otherimplantations. Likewise, these agonists such as adenosine A_(2A)receptor agonists may be used to increase bone mass or may ameliorateloss of bone mass in any of these conditions. It was determined thatadenosine A_(2A) receptor occupancy blocks osteoclast formation bymurine splenocytes incubated with macrophage colony stimulating factor(M-CSF) and receptor activator of NFkB ligand (RANKL) in vitro. Animalslacking adenosine A_(2A) receptors suffer from diminished bone mass invivo. These results suggest that adenosine A_(2A) receptor agonists maybe useful in the treatment of osteoporosis, prosthetic joint looseningand other conditions in which osteoclasts play a pathogenic role (e.g.Paget's Disease).

Adenosine, a potent endogenous physiological mediator, regulates a widevariety of physiological processes via interaction with one or more offour G protein-coupled receptors (A₁, A_(2A), A_(2B), and A₃), expressedon many cell types, including neutrophils, macrophages, fibroblasts, andendothelial cells. Because adenosine A_(2A) receptors inhibit theformation of giant cells from peripheral blood monocytes in vitro it wasdetermined that adenosine, acting through one or another of thesereceptors, regulated the formation of osteoclasts.

Osteoclast formation is reduced by adenosine A_(2A) receptor occupancysince pharmacologic blockade of these receptors completely inhibits theformation of osteoclasts in vitro.

In one embodiment, agents that interact with (e.g., bind to) and block,agonize or stimulate an adenosine receptor, in particular, A_(2A) (e.g.,a functionally active fragment), are identified in a cell-based assaysystem. In accordance with this embodiment, cells expressing anadenosine receptor, a fragment of an adenosine receptor, an adenosinereceptor related polypeptide, or a binding fragment thereof, arecontacted with a candidate compound or a control compound and theability of the candidate compound to interact with the receptor orfragment thereof is determined. Alternatively, the ability of acandidate compound to compete for binding with a known ligand orcompound known to bind the receptor is measured. If desired, this assaymay be used to screen a plurality (e.g. a library) of candidatecompounds. The cell, for example, can be of prokaryotic origin (e.g., E.coli) or eukaryotic origin (e.g., yeast, insect or mammalian). Further,the cells can express the receptor endogenously or be geneticallyengineered to express the receptor, a binding fragment or a receptorfusion protein. In some embodiments, the receptor or fragment thereof,or the candidate compound is labeled, for example with a radioactivelabel (such as ³²P, ³⁵S or ¹²⁵I) or a fluorescent label (such asfluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde or fluorescamine) to enable detecting aninteraction between the A_(2A) receptor and a candidate compound. Theability of the candidate compound to interact directly or indirectlywith a receptor or binding fragment thereof or a fusion protein or tomodulate the activity of the receptor can be determined by methods knownto those of skill in the art. For example, the interaction or modulationby a candidate compound can be determined by flow cytometry, ascintillation assay, immunoprecipitation or western blot analysis, basedon the present description, or by a competitive radioreceptor assay.

Selecting the compounds that interact with or bind to an adenosinereceptor or otherwise agonize or stimulate the receptor may be performedin multiple ways. The compounds may first be chosen based on theirstructural and functional characteristics, using one of a number ofapproaches known in the art. For instance, homology modeling can be usedto screen small molecule libraries in order to determine which moleculeswould be candidates to interact with the receptor thereby selectingplausible targets. The compounds to be screened can include both naturaland synthetic ligands. Furthermore, any desired compound may be examinedfor its ability to interact with or bind to the receptor.

Binding to or interaction with adenosine receptors may be determined byperforming an assay such as, for example, a binding assay between adesired compound and an adenosine receptor. In one aspect, this is doneby contacting said compound to an adenosine receptor and determining itsdissociation rate. Numerous possibilities for performing binding assaysare well known in the art. The indication of a compound's ability tobind to an adenosine receptor is determined, e.g., by a dissociationrate, and the correlation of binding activity and dissociation rates iswell established in the art. For example, the assay may be performed byradio-labeling a reference compound, or other suitable radioactivemarker, and incubating it with the cell bearing an adenosine receptor,in particular, A_(2A). Test compounds are then added to these reactionsin increasing concentrations. After optimal incubation, the referencecompound and receptor complexes are separated, e.g., with chromatographycolumns, and evaluated for bound ¹²⁵I-labeled peptide with a gamma (γ)counter. The amount of the test compound necessary to inhibit 50% of thereference compound's binding is determined. These values are thennormalized to the concentration of unlabeled reference compound'sbinding (relative inhibitory concentration(RIC)⁻¹=concentration_(test)/concentration_(reference)). A small RIC⁻¹value indicates strong relative binding, whereas a large RIC⁻¹ valueindicates weak relative binding. See, for example, Latek et al., Proc.Natl. Acad. Sci. USA, Vol. 97, No. 21, pp. 11460-11465, 2000. Anadenosine receptor agonist mimic may be computationally evaluated anddesigned by means of a series of steps in which chemical groups orfragments are screened and selected for their ability to associate withthe individual binding pockets or interface surfaces of the protein(e.g. the A_(2A) receptor). One skilled in the art may employ one ofseveral methods to screen chemical groups or fragments for their abilityto associate with the adenosine receptor. This process may begin byvisual inspection of, for example, the protein/protein interfaces or thebinding site on a computer screen based on the available crystal complexcoordinates of the receptor, including a protein known to interact withselected fragments or chemical groups may then be positioned in avariety of orientations, or docked, at an individual surface of thereceptor that participates in a protein/protein interface or in thebinding pocket. Docking may be accomplished using software such asQUANTA and SYBYL, followed by energy minimization and molecular dynamicswith standard molecular mechanics forcefields, such as CHARMM and AMBER(AMBER, version 4.0 (Kollman, University of California at San Francisco,copyright, 1994); QUANTA/CHARMM (Molecular Simulations, Inc.,Burlington, Mass., copyright, 1994)). Specialized computer programs mayalso assist in the process of selecting fragments or chemical groups.These include: GRID (Goodford, 1985, J. Med. Chem. 28:849-857),available from Oxford University, Oxford, UK; MCSS (Miranker & Karplus,1991, Proteins: Structure, Function and Genetics 11:29-34), availablefrom Molecular Simulations, Burlington, Mass.; AUTODOCK (Goodsell &Olsen, 1990, Proteins: Structure, Function, and Genetics 8:195-202),available from Scripps Research Institute, La Jolla, Calif.; and DOCK(Kuntz et al., 1982, J. Mol. Biol. 161:269-288), available fromUniversity of California, San Francisco, Calif. Once suitable chemicalgroups or fragments that bind to an adenosine receptor have beenselected, they can be assembled into a single compound or agonist.Assembly may proceed by visual inspection of the relationship of thefragments to each other in the three-dimensional image displayed on acomputer screen in relation to the structure coordinates thereof. Thiswould be followed by manual model building using software such as QUANTAor SYBYL. Useful programs to aid one of skill in the art in connectingthe individual chemical groups or fragments include: CAVEAT (Bartlett etal., 1989, ‘CAVEAT: A Program to Facilitate the Structure-Derived Designof Biologically Active Molecules’. In Molecular Recognition in Chemicaland Biological Problems’, Special Pub., Royal Chem. Soc. 78:182-196),available from the University of California, Berkeley, Calif.; 3DDatabase systems such as MACCS-3D (MDL Information Systems, San Leandro,Calif.). This area is reviewed in Martin, 1992, J. Med. Chem.35:2145-2154); and HOOK (available from Molecular Simulations,Burlington, Mass.). Instead of proceeding to build an adenosine receptoragonist mimic, in a step-wise fashion one fragment or chemical group ata time, as described above, such compounds may be designed as a whole or‘de novo’ using either an empty binding site or the surface of a proteinthat participates in protein/protein interactions or optionallyincluding some portion(s) of a known activator(s). These methodsinclude: LUDI (Bohm, 1992, J. Comp. Aid. Molec. Design 6:61-78),available from Molecular Simulations, Inc., San Diego, Calif.; LEGEND(Nishibata et al., 1991, Tetrahedron 47:8985), available from MolecularSimulations, Burlington, Mass.; and LeapFrog (available from Tripos,Inc., St. Louis, Mo.). Other molecular modeling techniques may also beemployed in accordance with this invention. See, e.g., Cohen et al.,1990, J. Med. Chem. 33:883-894. See also, Navia & Murcko, 1992, CurrentOpinions in Structural Biology 2:202-210.

Once a compound has been designed by the above methods, the efficiencywith which that compound may bind to or interact with the adenosinereceptor protein may be tested and optimized by computationalevaluation. Agonists may interact with the receptor in more than oneconformation that is similar in overall binding energy. In those cases,the deformation energy of binding is taken to be the difference betweenthe energy of the free compound and the average energy of theconformations observed when the inhibitor binds to the receptor protein.

A compound selected for binding to the adenosine receptor may be furthercomputationally optimized so that in its bound state it would preferablylack repulsive electrostatic interaction with the target protein. Suchnon-complementary electrostatic interactions include repulsivecharge-charge, dipole-dipole and charge-dipole interactions.Specifically, the sum of all electrostatic interactions between thecompound and the receptor protein when the mimic is bound to itpreferably make a neutral or favorable contribution to the enthalpy ofbinding. Specific computer software is available in the art to evaluatecompound deformation energy and electrostatic interaction. Examples ofprograms designed for such uses include: Gaussian 92, revision C(Frisch, Gaussian, Inc., Pittsburgh, Pa. copyright 1992); AMBER, version4.0 (Kollman, University of California at San Francisco, copyright1994); QUANTA/CHARMM (Molecular Simulations, Inc., Burlington, Mass.,copyright 1994); and Insight II/Discover (Biosym Technologies Inc., SanDiego, Calif., copyright 1994). These programs may be implemented, forinstance, using a computer workstation, as are well-known in the art.Other hardware systems and software packages will be known to thoseskilled in the art.

Once an adenosine receptor modulating compound, preferably an agonist,has been optimally designed, for example as described above,substitutions may then be made in some of its atoms or chemical groupsin order to improve or modify its binding properties, or itspharmaceutical properties such as stability or toxicity. Generally,initial substitutions are conservative, i.e., the replacement group willhave approximately the same size, shape, hydrophobicity and charge asthe original group. Substitutions known in the art to alter conformationshould be avoided. Such altered chemical compounds may then be analyzedfor efficiency of binding to the receptor by the same computer methodsdescribed in detail above.

Candidate Compounds and Agents

Examples of agents, candidate compounds or test compounds include, butare not limited to, nucleic acids (e.g., DNA and RNA), carbohydrates,lipids, proteins, peptides, peptidomimetics, small molecules and otherdrugs. In one preferred aspect, agents can be obtained using any of thenumerous suitable approaches in combinatorial library methods known inthe art, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the “one-bead one-compound” library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, 1997, Anticancer Drug Des.12:145; U.S. Pat. No. 5,738,996 and U.S. Pat. No. 5,807,683).

Phage display libraries may be used to screen potential ligands oradenosine receptor modulators. Their usefulness lies in the ability toscreen, for example, a library displaying a large number of differentcompounds. For use of phage display libraries in a screening process,see, for instance, Kay et al., Methods, 240-246, 2001. An exemplaryscheme for using phage display libraries to identify compounds that bindor interact with an adenosine receptor may be described as follows:initially, an aliquot of the library is introduced into microtiter platewells that have previously been coated with target protein, e.g. A_(2A)receptor. After incubation (e.g., 2 hours), the nonbinding phage arewashed away, and the bound phage are recovered by denaturing ordestroying the target with exposure to harsh conditions such as, forinstance pH 2, but leaving the phage intact. After transferring thephage to another tube, the conditions are neutralized, followed byinfection of bacteria with the phage and production of more phageparticles. The amplified phage are then rescreened to complete one cycleof affinity selection. After three or more rounds of screening, thephage are plated out such that there are individual plaques that can befurther analyzed. For example, the conformation of binding activity ofaffinity-purified phage for the adenosine A_(2A) receptor may beobtained by performing ELISAs. One skilled in the art can easily performthese experiments. In one aspect, an A_(2A) receptor molecule used forany of the assays may be a recombinant A_(2A) receptor protein, or anA_(2A) fusion protein, an analog, derivative, or mimic thereof.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad.Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al., 1993,Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl.33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al., 1994, J. Med. Chem. 37:1233.

Libraries of compounds may be presented, e.g., in solution (Houghten,1992, Bio/Techniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA89:1865-1869) or phage (Scott and Smith, 1990, Science 249:386-390;Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici, 1991, J. Mol. Biol.222:301-310).

The methods of screening compounds may also include the specificidentification or characterization of such compounds, whose effect onbone resorption was determined by the methods described above. If theidentity of the compound is known from the start of the experiment, noadditional assays are needed to determine its identity. However, if thescreening for compounds that modulate the adenosine A_(2A) receptor isdone with a library of compounds, it may be necessary to performadditional tests to positively identify a compound that satisfies allrequired conditions of the screening process. There are multiple ways todetermine the identity of the compound. One process involves massspectrometry, for which various methods are available and known to theskilled artisan (e.g. the neogenesis website). Neogenesis' ALIS(automated ligand identification system) spectral search engine and dataanalysis software allow for a highly specific identification of a ligandstructure based on the exact mass of the ligand. One skilled in the artcan also readily perform mass spectrometry experiments to determine theidentity of the compound.

Antibodies, including polyclonal and monoclonal antibodies, particularlyanti-A_(2A) receptor antibodies and neutralizing antibodies may beuseful as compounds to modulate osteoclast differentiation and/orfunction. These antibodies are available from such vendors as UpstateBiologicals, Santa Cruz, or they made be prepared using standardprocedures for preparation of polyclonal or monoclonal antibodies knownto those skilled in the art. Also, antibodies including both polyclonaland monoclonal antibodies, and drugs that modulate the activity of theadenosine receptor and/or its subunits may possess certain diagnosticapplications and may for example, be utilized for the purpose ofdetecting and/or measuring conditions such as bone diseases, bone loss,or osteoclast differentiation and/or function. The adenosine receptor orits subunits may be used to produce both polyclonal and monoclonalantibodies to themselves in a variety of cellular media, by knowntechniques such as the hybridoma technique utilizing, for example, fusedmouse spleen lymphocytes and myeloma cells. Likewise, small moleculesthat mimic or act as agonists for the activities of the A_(2A) receptormay be discovered or synthesized, and may be used in diagnostic and/ortherapeutic protocols.

Therapeutic and Prophylactic Compositions and their Use

Candidates for therapy with the agents identified by the methodsdescribed herein are patients either suffering from bone resorption orpatients who have a medical prosthesis implanted or who contemplatereceiving an implant medical prosthetic device.

The invention provides methods of treatment featuring administering to asubject an effective amount of an agent of the invention. The compoundis preferably substantially purified (e.g., substantially free fromsubstances that limit its effect or produce undesired side-effects). Thesubject is preferably an animal, including but not limited to animalssuch as monkeys, cows, pigs, horses, chickens, cats, dogs, etc., and ispreferably a mammal, and most preferably human. In one specificembodiment, a non-human mammal is the subject. In another specificembodiment, a human mammal is the subject. Accordingly, the agentsidentified by the methods described herein may be formulated aspharmaceutical compositions to be used for prophylaxis or therapeuticuse to treat these patients.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, or microcapsules. Methods of introduction can be enteralor parenteral and include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, topical and oral routes. The compounds may be administered byany convenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compositions of the invention into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection; intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent. In a specific embodiment, it may be desirable toadminister the pharmaceutical compositions of the invention locally tothe area in need of treatment.

Such compositions comprise a therapeutically effective amount of anagent, and a pharmaceutically acceptable carrier. In a particularembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the subject. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

In another embodiment, the compound can be delivered in a vesicle, inparticular a liposome (Langer (1990) Science 249:1527-1533; Treat etal., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327)

In yet another embodiment, the compound can be delivered in a controlledor sustained release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201;Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J.Med. 321:574). In another embodiment, polymeric materials can be used(See, Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger et al., (1983) Macromol. Sci.Rev. Macromol. Chem. 23:61; Levy et al. (1985) Science 228:190; Duringet al. (1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg.71:105). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the subject bone orprosthesis, thus requiring only a fraction of the systemic dose (see,e.g., Goodson, in Medical Applications of Controlled Release (1984)supra, vol. 2, pp. 115-138). Other suitable controlled release systemsare discussed in the review by Langer (1990) Science 249:1527-1533.

The present invention further contemplates therapeutic compositionsuseful in practicing the therapeutic methods of this invention. Asubject therapeutic composition includes, in admixture, apharmaceutically acceptable excipient (carrier) and one or more of anadenosine receptor modulator, such as an adenosine A_(2A) receptoragonist, as described herein as an active ingredient. In a preferredembodiment, the composition comprises one or more compounds or agentscapable of mimicking or serving as an agonist for the adenosine A_(2A)receptor.

Effects of the compounds or agents of the invention can first be testedfor their ability to stimulate or mimic the adenosine receptor usingstandard techniques known in the art. More particularly, the selectivityof the compounds for the receptor can be assessed using radioligandbinding assays whereby a test or candidate compound can be assayed forits ability to bind to a cell having or expressing the receptor(including any of the known adenosine receptors, A₁, A_(2A), A_(2B) orA₃). Cells can be transfected with the nucleic acid encoding the variousadenosine receptors and competitive binding assays with radiolabeledligands run to evaluate the specificity of the particular candidatecompounds. The cDNAs for human A₁ (see GenBank accession numberBC026340), A_(2A) (see GenBank accession number NM₀₀₀₆₇₅), A_(2B) (seeGenBank accession number NM₀₀₀₆₇₆) or A₃ (see GenBank accession numberAY136749 or L22607 or NM₀₀₀₆₇₇) can be used to prepare the nucleic acidconstructs for use in these methods.

The present compounds or agents that modulate the adenosine receptor, inparticular, the agonists of the A_(2A) receptor, themselves can be usedas the sole active agents, or can be used in combination with one ormore other active ingredients. In particular, combination therapy usingthe adenosine receptor agonists with one or more other agents iscontemplated. These agents are known in the art, and can be selectedfrom anti-inflammatory compounds, bisophosphonates, soluble RANK, andbone morphogenetic proteins, for instance.

When contemplating combination therapy with an adenosine receptoragonist and one or more of the above-noted agents, it is important toassess clinical safety by methods known to those skilled in the art.Appropriate dose titration may be necessary when certain groups ofcompounds are contemplated for use together.

The compounds or compositions of the invention may be combined foradministration with or embedded in polymeric carrier(s), biodegradableor biomimetic matrices or in a scaffold. The carrier, matrix or scaffoldmay be of any material that will allow composition to be incorporatedand expressed and will be compatible with the addition of cells or inthe presence of cells. Preferably, the carrier matrix or scaffold ispredominantly non-immunogenic and is biodegradable. Examples ofbiodegradable materials include, but are not limited to, polyglycolicacid (PGA), polylactic acid (PLA), hyaluronic acid, catgut suturematerial, gelatin, cellulose, nitrocellulose, collagen, albumin, fibrin,alginate, cotton, or other naturally-occurring biodegradable materials.It may be preferable to sterilize the matrix or scaffold material priorto administration or implantation, e.g., by treating it with ethyleneoxide or by gamma irradiation or irradiation with an electron beam. Inaddition, a number of other materials may be used to form the scaffoldor framework structure, including but not limited to: nylon(polyamides), dacron (polyesters), polystyrene, polypropylene,polyacrylates, polyvinyl compounds (e.g., polyvinylchloride),polycarbonate (PVC), polytetrafluorethylene (PTFE, teflon), thermanox(TPX), polymers of hydroxy acids such as polylactic acid (PLA),polyglycolic acid (PGA), and polylactic acid-glycolic acid (PLGA),polyorthoesters, polyanhydrides, polyphosphazenes, and a variety ofpolyhydroxyalkanoates, and combinations thereof. Matrices suitableinclude a polymeric mesh or sponge and a polymeric hydrogel. In thepreferred embodiment, the matrix is biodegradable over a time period ofless than a year, more preferably less than six months, most preferablyover two to ten weeks. The polymer composition, as well as method ofmanufacture, can be used to determine the rate of degradation. Forexample, mixing increasing amounts of polylactic acid with polyglycolicacid decreases the degradation time. Meshes of polyglycolic acid thatcan be used can be obtained commercially, for instance, from surgicalsupply companies (e.g., Ethicon, N.J.). A hydrogel is defined as asubstance formed when an organic polymer (natural or synthetic) iscross-linked via covalent, ionic, or hydrogen bonds to create athree-dimensional open-lattice structure which entraps water moleculesto form a gel. In general, these polymers are at least partially solublein aqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof.

For use in treating animal subjects, the compositions of the inventioncan be formulated as pharmaceutical or veterinary compositions.Depending on the subject to be treated, the mode of administration, andthe type of treatment desired, e.g., prevention, prophylaxis, therapy;the compositions are formulated in ways consonant with these parameters.A summary of such techniques is found in Remington's PharmaceuticalSciences, latest edition, Mack Publishing Co., Easton, Pa.

The preparation of therapeutic compositions containing small organicmolecules polypeptides, analogs or active fragments as activeingredients is well understood in the art. The compositions of thepresent invention may be administered parenterally, orally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques. Formulations may beprepared in a manner suitable for systemic administration or for topicalor local administration. Systemic formulations include, but are notlimited to those designed for injection (e.g., intramuscular,intravenous or subcutaneous injection) or may be prepared fortransdermal, transmucosal, nasal, or oral administration. Suchcompositions may be prepared as injectables, either as liquid solutionsor suspensions, however, solid forms suitable for solution in, orsuspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. The formulation will generally include a diluentas well as, in some cases, adjuvants, buffers, preservatives and thelike. In addition, if desired, the composition can contain minor amountsof auxiliary substances such as wetting or emulsifying agents, pHbuffering agents which enhance the effectiveness of the activeingredient.

A small organic molecule/compound, a polypeptide, an analog or activefragment thereof can be formulated into the therapeutic composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide or antibody molecule) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like. For oral administration, thecompositions can be administered also in liposomal compositions or asmicroemulsions. Suitable forms include syrups, capsules, tablets, as isunderstood in the art.

The compositions of the present invention may also be administeredlocally to sites in subjects, both human and other vertebrates, such asdomestic animals, rodents and livestock, using a variety of techniquesknown to those skilled in the art. For example, these may includesprays, lotions, gels or other vehicles such as alcohols, polyglycols,esters, oils and silicones.

The administration of the compositions of the present invention may bepharmacokinetically and pharmacodynamically controlled by calibratingvarious parameters of administration, including the frequency, dosage,duration mode and route of administration. Variations in the dosage,duration and mode of administration may also be manipulated to producethe activity required.

The therapeutic adenosine receptor modulator (e.g. inhibitor)compositions are conventionally administered in the form of a unit dose,for instance intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionof the present invention refers to physically discrete units suitable asunitary dosage for humans, each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effectin association with the required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the agentselected for treating the subject, the dosage formulation, and in atherapeutically effective amount. If one desires to achieve the desiredeffect in vitro, the effective amounts may range from about 0:1 nM toabout 10 μM, more preferably about 0.1 nM to about 5 μM, and mostpreferably from about 0.1 nM to about 1 nM. The desired effect refers tothe effect of the agent on reducing or inhibiting osteoclastdifferentiation or stimulation, reducing or inhibiting bone resorptionand reducing or inhibiting loosening of a medical prosthesis. Moreover,the quantity of the adenosine receptor agonist to be administereddepends on the subject to be treated, and degree of stimulation ormimicry of the adenosine receptor desired or the extent or severity ofbone resorption. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner and are peculiarto each individual. However, suitable dosages to achieve the desiredtherapeutic effect in vivo may range from about 0.1 mg/kg body weightper day to about 200 mg/kg body weight per day, or from about 1.0 mg/kgbody weight per day to about 100 mg/kg body weight per day, preferablyabout 25 mg/kg body weight per day to about 50 mg/kg body weight perday. In a particular embodiment, the term “about” means within 20%,preferably within 10%, and more preferably within 5%. The preferred dosewill depend on the route of administration. However, dosage levels arehighly dependent on the nature of the disease or situation, thecondition of the subject, the judgment of the practitioner, and thefrequency and mode of administration. If the oral route is employed, theabsorption of the substance will be a factor effecting bioavailability.A low absorption will have the effect that in the gastro-intestinaltract higher concentrations, and thus higher dosages, will be necessary.Suitable regimes for initial administration and further administrationare also variable, but are typified by an initial administrationfollowed by repeated doses at one or more hour intervals by a subsequentinjection or other administration. Alternatively, continuous intravenousinfusion sufficient to maintain desired concentrations, e.g. in theblood, are contemplated. The composition may be administered as a singledose multiple doses or over an established period of time in aninfusion.

It will be understood that the appropriate dosage of the substanceshould suitably be assessed by performing animal model tests, where theeffective dose level (e.g., ED₅₀) and the toxic dose level (e.g. TD₅₀)as well as the lethal dose level (e.g. LD₅₀ or LD₁₀) are established insuitable and acceptable animal models. Further, if a substance hasproven efficient in such animal tests, controlled clinical trials shouldbe performed.

The compounds or compositions of the present invention may be modifiedor formulated for administration at the site of pathology. Suchmodification may include, for instance, formulation which facilitate orprolong the half-life of the compound or composition, particularly inthe environment. Additionally, such modification may include theformulation of a compound or composition to include a targeting proteinor sequence which facilitates or enhances the uptake of thecompound/composition to bone or bone precursor cells. In a particularembodiment, such modification results in the preferential targeting ofthe compound to bone or bone precursor cells versus other locations orcells. In one embodiment, a tetracycline, tetracycline family orbisphosphonate may be utilized to target the compound or composition ofthe present invention to bone or bone cells, including osteoclasts andosteoclast precursors. Novel heterocycles as bone targeting compoundsare disclosed in U.S. Patent Publication No. 2002/0103161 A₁, which isincorporated herein by reference in its entirety.

Pharmaceutically acceptable carriers useful in these pharmaceuticalcompositions include, e.g., ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Sterile injectable forms of the compositions may be aqueous oroleaginous suspensions. The suspensions may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

Parenteral formulations may be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionsmay be administered once a day or on an “as needed” basis.

The pharmaceutical compositions may be orally administered in any orallyacceptable dosage form including, capsules, tablets, aqueous suspensionsor solutions. In the case of tablets for oral use, carriers commonlyused include lactose and corn starch. Lubricating agents, such asmagnesium stearate, are also typically added. For oral administration ina capsule form, useful diluents include lactose and dried cornstarch.When aqueous suspensions are required for oral use, the activeingredient is combined with emulsifying and suspending agents. Ifdesired, certain sweetening, flavoring or coloring agents may also beadded.

Alternatively, the pharmaceutical compositions may be administered inthe form of suppositories for rectal administration. These can beprepared by mixing the agent with a suitable non-irritating excipientwhich is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum to release the drug. Such materialsinclude cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically. Topical application can be effected in a rectalsuppository formulation (see above) or in a suitable enema formulation.Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, mineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, thepharmaceutical compositions can be formulated in a suitable lotion orcream containing the active components suspended or dissolved in one ormore pharmaceutically acceptable carriers. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcoholand water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects (a) approval by the agency ofmanufacture, use or sale for human administration, (b) directions foruse, or both.

The invention also provides prosthetic devices having an adenosinereceptor modulating agent such as an adenosine A_(2A) receptor agonistthereon or therein. The adenosine receptor modulating agent such as anadenosine A_(2A) receptor agonist may be present in a compositionapplied to one or more surfaces of the prosthetic device or theadenosine receptor modulating agent such as an adenosine A_(2A) receptoragonist may be present within the prosthetic device. That is, theadenosine receptor modulating agent such as an adenosine A_(2A) receptoragonist, may be present within the very matrix of the prosthetic devicesuch as for instance within the cement, e.g. methylmethacrylate cementthat forms the prosthetic device.

Effective Doses

Toxicity and therapeutic efficacy of compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage tounaffected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a dose range for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage can varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any compound used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC₅₀(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to optimize efficacious doses for administrationto humans. Plasma levels can be measured by any technique known in theart, for example, by high performance liquid chromatography.

In addition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each subject's circumstances.Normal dose ranges used for particular therapeutic agents employed forspecific diseases can be found in the Physicians' Desk Reference 54^(th)Edition (2000).

EXAMPLES

The following examples are set forth to provide those of ordinary skillin the art with a description of how to make and use the methods andcompositions of the invention, and are not intended to limit the scopethereof. Efforts have been made to insure accuracy of numbers used(e.g., amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is average molecular weight,temperature is in degrees Centigrade, and pressure is at or nearatmospheric.

Example 1

Bone mineral density in 6-month old A_(2A) receptor knockout mice andotherwise genetically identical wild type mice was determined. TheA_(2A) receptor knockout mice had significantly lower bone mineraldensity, and examination of their long bones showed increased boneresorption and increased numbers of osteoclasts. Electron microscopydemonstrated that the osteoclasts appeared to be much more active inbone resorption than in the wild type mice. When studied in vitroRAW264.7 cells, a murine cell line, can be induced to differentiate intoosteoclast-like cells. Culture of these cells in the presence of anadenosine A_(2A) receptor antagonist inhibited osteoclast formation.Bone mineral density was determined in anesthetized mice by use ofquantitative Xray densitometry (DEXA scan, Piximus GE).

Electron Microscopy of Osteoclasts

Both tibiae and femora of five animals will be fixed in 2.5%glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 12 hoursat ambient temperature. After being rinsed three times for 20 min in thesame buffer, the material will be postfixed for 6 hours in 1% osmiumtetroxide (in 0.1 M sodium cacodylate buffer), dehydrated in acetone andembedded. Sections are prepared, stained with Cathepsin K and examinedin the electron microscope. The thin sections are scanned systematicallyand all osteoclasts encountered are photographed.

Characterization of Osteoclasts

Osteoclast formation was evaluated by quantification of TRAP-positiveMNCs as described previously. TRAP is preferentially expressed at highlevels in osteoclasts and is considered, especially in the mouse, to bean osteoclast marker. After incubation, cells on wells were washed inPBS, fixed in 4% paraformaldehyde for 10 min, and stained for acidphosphatase in the presence of 0.3 M sodium tartrate (Sigma-Aldrich).The substrate used was napthol AS-BI phosphate (Sigma-Aldrich). Onlythose cells that were strongly TRAP-positive (dark red) and have morethan 3 nuclei were counted by light microscopy. To evaluate the size ofthe Osteoclast over time, we counted the number of nuclei per OCL in 50OCLs for each of the three wells(n=150). The mean number of nuclei perOCL (MNN) and SE were calculated for each time-point, and ANOVA analysiswas performed to determine statistically significant differences.

Example 2

A model of prosthesis loosening and bone resorption will be established.A subcutaneous air pouch will be created on the dorsum of a mouse. Ultrahigh molecular weight polyethylene debris will be implanted into the airpouch followed by implantation of calvaria from syngeneic mice. Thematerial will be harvested from the mice after 14 days, and boneresorption and inflammation will be assessed. An adenosine A_(2A)receptor agonist (CGS21680) will be mixed into methylmethacrylate glueapplied to the calvarial bone in some animals or methylmethacrylate gluealone applied to the calvarial bone in controls. Numbers of TRAP+osteoclasts, bone resorption (microCT), bone collagen content (vonGieson stain), inflammatory cytokine production (IL-1, TNF, IL-6) inexudates (ELISA) and by FISH in bone will be assessed according to theprocedures described by Ren, et al., 2006, Journal of OrthopaedicResearch 24:1575-1586.

Similar preparations in adenosine A_(2A) receptor knockout mice willalso be studied to confirm the specificity of the effect. The data willdemonstrate that adenosine A_(2A) receptor agonists can be used toprevent bone resorption in a simple model of prosthesis loosening. Thedata will further confirm that A_(2A) receptor agonists may be used forpreventing peri-prosthetic bone resorption. Currently two adenosineA_(2A) receptor agonists are in testing for topical application forwound healing (King, currently in Phase II trials) and as a coronaryvasodilator for pharmacologic stress testing (ATL, Inc. andBristol-Myers-Squibb, Phase I recently reported). Both agents areextremely short-lived and relatively non-toxic.

Example 3

Background.

Osteoclasts are bone-resorbing, multinucleated giant cells that areessential for bone remodeling and that are formed through the fusion ofmononuclear precursor cells. Osteoclasts differentiate from hemopoieticprecursors of the monocyte/macrophage lineage in the presence of M-CSFand receptor activator of NF-kB ligand (RANKL). Deficiency ofosteoclasts leads to osteopetrosis, a condition characterized byincreased bone density. Nonetheless, most bone diseases are due toincreased bone resorption by osteoclasts and inhibition ofosteoclast-mediated bone resorption is a primary therapeutic objective.Indeed, most current therapies for osteoporosis are directed atinhibition of osteoclast function. Adenosine, a potent endogenousphysiological mediator, regulates a wide variety of physiologicalprocesses via interaction with one or more of four G protein-coupledreceptors (A₁, A_(2A), A_(2B) and A₃), expressed on many cell types.Because we have previously reported that adenosine A_(2A) receptoroccupancy is required for fusion of stimulated human monocytes to formgiant cells in vitro we determined whether there was a similarrequirement for A_(2A) receptor occupancy in osteoclast formation andfunction in vitro and in vivo models.

Materials and Methods

RAW264.7 is a macrophage cell line having the capacity to formosteoclast-like cells. Because the RAW264.7 cells provide an excellentin vitro model for dissecting the cellular and molecular regulation ofosteoclast differentiation and activation, we studied the effect ofadenosine A_(2A) receptors on the osteoclasts generated from theRAW264.7 cells.

Total RNA Extraction/RT-PCR from RAW264.7, Splenocyte and Bone MarrowCells.

After the cultured cells became confluent in tissue culture flasks andwere passaged three times, the cell suspensions were lysed in Trizolreagent (Invitrogen Life Technologies, UK), and total RNA from dissolvedspecimens was extracted according to the manufacturer's instructions.First, single stranded complementary DNA (cDNA) was synthesized fromtotal RNA from each sample using a cDNA synthesis kit (Invitrogen LifeTechnologies, UK). cDNA was then amplified by polymerase chain reaction(PCR) to generate products corresponding to mRNA encoding mice geneproducts for adenosine receptors A₁, A_(2A), A_(2B) and A₃ and GAPDHmRNAs were assessed by RT-PCR.

Cell Culture and Osteoclastogenesis.

Raw264.7 cells were cultured in 24 well plates (5×10⁴/ml) with α-MEM and10% heat inactivated Fetal Bovine Serum (FBS). Cells were then incubatedwith 30 ng/ml of recombinant mouse RANKL in the presence of eitherA_(2A) agonist (CGS 21680) or antagonist (ZM241385). After four days ofculture, cells were fixed with 10% formalin and stained fortartrate-resistant acid phosphatase (TRAP). Osteoclasts were identifiedas TRAP-positive cells with 3 or more nuclei. The number ofTRAP-positive multinucleated cells/well was then enumerated.

Western Blotting.

Osteoclast precursors were preincubated with or without CGS21680 (1μg/ml) or ZM241385 (1 μg/ml) for 4 days and then stimulated with RANKL(30 ng/ml) for the indicated period. For isolation of total proteins,cells were washed twice with PBS and lysed in RIPA buffer. Proteins wereestimated and boiled with sample buffer for 5 min and were subjected toelectrophoresis on 10% SDS-PAGE. Proteins were transferred onto anitrocellulose membrane. Membranes were blocked for 2 hours in blockingsolution (5% nonfat dry milk in TBS containing 0.1% Tween 20) andexposed to primary Abs overnight at 4° C. After washing, the membraneswere incubated for 1 hour at room temperature with secondary Ab, and theproteins were detected using ECL reagents according to themanufacturer's instructions. To reprobe the membranes with other Abs,the membranes were stripped with 100 mM 2-ME, 2% SDS, and 62.5 mMTris-HCl (pH 6.9) for 20 min at 50° C., followed by immunoblotting asdescribed above.

Bone Density Measurements.

In vivo bone density measurements in A_(2A) WT (wild type) and A_(2A) KO(knock out) mice were performed by dual energy x-ray absorptiometry,using a PIXImus densitometer (Lunar Corp., Madison, Wis.). Measurementswere made at 6 months of age. Anesthetized mice (30 mg ketamine/kg bodywt and 3 mg xylazine/kg body wt, ip) were placed in the prone position,and scans were performed and an acquisition time of 5 min.

Micro-X-Ray Computed Tomography (μCT) Analysis of Bone Mass.

For measurements of the bone volume (trabecular bone volume (BV/TV)),the femurs from four A_(2A) WT and 4 A_(A2A) KO mice were subjected toμCT analysis with MS-8 (MS-8, GE Healthcare, London, Ontario, Canada) at18 μm isotropic resolution—scans calibrated by air, water and a mineralstandard material phantom/Parker algorithm for digital reconstruction.The fractional bone volume (BV/TV) was measured in a cortical—rectanglearea in the proximal diaphysis (1.8 mm) parts.

Electron Microscopy of Osteoclasts.

Femurs of five animals will be fixed in 2.5% glutaraldehyde in 0.1 Msodium cacodylate buffer (pH 7.4) for 12 hours at ambient temperature.After being rinsed three times for 20 min in the same buffer, thematerial will be postfixed for 6 hours in 1% osmium tetroxide (in 0.1 Msodium cacodylate buffer), dehydrated in acetone and embedded. Sectionswill be prepared, stained with Cathepsin K and examined in an electronmicroscope.

Results

Osteoclasts Express mRNA and Protein for all Four Adenosine Receptors.

It was determined whether osteoclasts derived from bone marrowosteoclast precursors, splenocyte osteoclast precursors or the murinecell line RAW264.7 which differentiates into osteoclast like cells,express adenosine receptors. We initially measured mRNA levels forspecific adenosine receptors in each of the cells and cell lines. Asshown in FIG. 1A, all of these cells and cell lines express message forall four adenosine receptors. As shown in FIG. 1B, Western Blot analysisof bone marrow-derived osteoclast lysates demonstrates the presence ofall four adenosine receptors.

Adenosine A_(2A) Receptor Knockout Mice have Diminished Bone Density andBone Mineral Ca⁺⁺.

As shown in FIG. 2, both bone mineral density and bone mineral Ca⁺⁺ arediminished in adenosine A_(2A) receptor knockout mice. These findingssuggest that adenosine receptor knockout mice either have increased boneabsorption or diminished bone production.

Adenosine A_(2A) Receptor Knockout Mice have Evidence for Increased BoneResorption and Increased Numbers of Osteoclasts in their Bones.

Alcian Blue staining of decalcified bone from WT (wild type) andadenosine A_(2A) knockout mice demonstrates a marked increase in thenumber of osteoclasts and diminished bone substance in the adenosineA_(2A) receptor knockout mice (FIG. 3).

The Increased Number of Osteoclasts Correlates with Diminished BoneMineral Density.

The increased number of osteoclasts present in the bones of A_(2A) KOmice, as compared to WT mice, correlates with the diminished bonemineral density observed by DEXA scan of these mic (FIG. 4).

Electron Microscopy Demonstrates Greater Apparent Activity ofOsteoclasts in A_(2A) KO Mice.

Examination of bone by electron microscopy demonstrates larger boneresorption pits (clear areas) in the bones of A_(2A) receptor knockoutmice than wild type mice (FIG. 5).

MicroCT of Bones from A_(2A) Knockout Mice Demonstrates DiminishedCortical and Trabecular Bone in the KO Mice as Compared to WT Mice.

By all measures of bone substance, there is a reduction in both corticaland trabecular bone in the femurs of A_(2A) receptor knockout (A₂ KO)mice as compared to their wild type littermates (WT) (Table 1).

Quantitative Micro CT Analysis of the Proximal Femurs of 4-Month-Old WTand A_(2A) KO Mice

TABLE 1 WT A₂KO t-test Bone Volume/Tissue 22.61 ± 2.154 N = 3 17.49 ±0.98 0.04 Volume BV/TV N = 3 Trabecular number 5.8 ± 0.07 N = 3 4.4 ±0.15 N = 3 0.0006 (Tb. N.) Trabecular separation 0.13 ± 0.004 N = 3 0.19± 0.006 0.001 (Tb. Sep.) N = 3 Cortical area 0.68 ± 0.04 N = 3 0.6 ±0.016 N = 3 0.05 Total area 1.5 ± 0.03 N = 3 1.3 ± 0.02 N = 3 0.02 BMC0.01 ± 0.0008 N = 3 0.01 ± 0.0003 0.06 N = 3 Outer Perimeter 4.6 ± 0.04N = 3 4.4 ± 0.04 N = 3 0.006 TMC 1.9 ± 0.12 N = 3 1.7 ± 0.05 N = 3 0.05

Adenosine A_(2A) Receptor Blockade Diminishes Osteoclast Formation InVitro by RAW264.7 Cells Induced to Undergo Osteoclast Differentiation.

To better understand why adenosine A_(2A) receptor knockout mice havediminished bone density as compared to wild type mice, the effect of anadenosine A_(2A) receptor antagonist on osteoclast formation wasdetermined. As shown in FIG. 6 adenosine A_(2A) receptor antagonisttreated RAW264.7 cells form an increased number of multinucleated TRAP+osteoclasts in culture than untreated RAW264.7 cells.

Adenosine A_(2A) Receptor Blockade Downregulates TRAF6 Protein inRAW264.7 Cells.

It was previously demonstrated that A₁ adenosine receptor blockadediminishes TRAF6 protein, a critical signaling protein for osteoclastdifferentiation, in bone marrow osteoclasts and RAW264.7 cells. Todetermine whether adenosine A_(2A) receptors regulate osteoclastdifferentiation by a similar mechanism, TRAF6 levels in RAW264.7 cellstreated with an adenosine A₁ receptor antagonist, an A_(2A) agonist andantagonist were determined. As shown in FIG. 7, both the A₁ receptorantagonist and A_(2A) receptor agonist diminish TRAF6 levels in RAW264.7cells.

Summary.

All adenosine receptor subtypes (A₁, A_(2A), A_(2B), A₃) are expressedin RAW264.7 cells and RAW264.7 derived osteoclasts. Blockade ofadenosine A_(2A) receptors enhances osteoclast formation in culturedRAW264.7 cells. Blockade of adenosine A_(2A) receptors enhancesosteoclast formation and function in the mouse model. Adenosine A_(2A)receptor knockout mice have osteoporosis. These results demonstrate thatadenosine A_(2A) receptors regulate bone homeostasis and serve astargets for drugs that prevent bone loss.

1. A method for inhibiting bone resorption comprising administering to asubject a therapeutically effective amount of an adenosine receptoragonist, or an analog, derivative or combination thereof.
 2. The methodof claim 1 wherein the adenosine receptor is selected from the groupconsisting of A₁, A_(2A), A_(2B) and A₃.
 3. The method of claim 2wherein the adenosine receptor is an A_(2A) receptor.
 4. The method ofclaim 1 wherein and the agonist is an adenosine receptor A_(2A) agonist.5. The method of claim 4 wherein and the adenosine receptor A_(2A)agonist is a selective adenosine receptor agonist.
 6. The method ofclaim 4 wherein the adenosine A_(2A) receptor agonist is administeredvia an implanted device.
 7. The method of claim 4 wherein the adenosineA_(2A) receptor agonist is selected from the group consisting of CGS21680, IB-MECA and R-PIA.
 8. The method of claim 4 wherein the adenosineA_(2A) receptor agonist is administered in combination with one or moreof other compounds or agents for inhibiting bone resorption, osteoclastdifferentiation and stimulation and prosthesis loosening or for reducinginflammation.
 9. A method for inhibiting differentiation and stimulationof osteoclasts comprising administering to a subject a therapeuticallyeffective amount of an adenosine receptor agonist, or an analog,derivative or combination thereof.
 10. The method of claim 9 wherein theadenosine receptor is selected from the group consisting of A₁, A_(2A),A_(2B) and A₃.
 11. The method of claim 10 wherein the adenosine receptoris an A_(2A) receptor
 12. The method of claim 9 wherein and the agonistis an adenosine receptor A_(2A) agonist.
 13. The method of claim 12wherein and the adenosine receptor A_(2A) agonist is a selectiveadenosine receptor agonist.
 14. The method of claim 12 wherein theadenosine A_(2A) receptor agonist is administered via an implanteddevice.
 15. The method of claim 12 wherein the adenosine A_(2A) receptoragonist is selected from the group consisting of CGS 21680, IB-MECA andR-PIA.
 16. The method of claim 12 wherein the adenosine A_(2A) receptoragonist is administered in combination with one or more of othercompounds or agents for inhibiting bone resorption, osteoclastdifferentiation and stimulation and prosthesis loosening or for reducinginflammation.
 17. A method of inhibiting or reducing loosening of amedical prosthesis comprising administering to the subject an amount ofan adenosine receptor agonist effective to inhibit or reduce boneresorption, or an analog, derivative or combination thereof.
 18. Themethod of claim 17 wherein the adenosine receptor is selected from thegroup consisting of A₁, A_(2A), A_(2B) and A₃.
 19. The method of claim18 wherein the adenosine receptor is an A_(2A) receptor
 20. The methodof claim 17 wherein and the agonist is an adenosine receptor A_(2A)agonist.
 21. The method of claim 20 wherein and the adenosine receptorA_(2A) agonist is a selective adenosine receptor agonist.
 22. The methodof claim 20 wherein the adenosine A_(2A) receptor agonist isadministered via an implanted device.
 23. The method of claim 20 whereinthe adenosine A_(2A) receptor agonist is selected from the groupconsisting of CGS 21680, IB-MECA and R-PIA.
 24. The method of claim 20wherein the adenosine A_(2A) receptor agonist is administered incombination with one or more of other compounds or agents for inhibitingbone resorption, osteoclast differentiation and stimulation andprosthesis loosening or for reducing inflammation.
 25. A prostheticdevice comprising an adenosine receptor agonist in an amount sufficientto inhibit osteoclast differentiation and stimulation, bone resorption,or loosening of a prosthetic device.
 26. The prosthetic device of claim25 wherein the adenosine receptor agonist is an adenosine A_(2A)receptor agonist.
 27. The prosthetic device of claim 25 wherein theadenosine receptor agonist is present within the prosthetic device. 28.A pharmaceutical composition comprising the adenosine receptor agonistalone or in combination with one or more compounds or agents effectivefor inhibiting inhibiting bone resorption, osteoclast differentiationand stimulation and the loosening of medical prostheses.
 29. Thepharmaceutical composition of claim 28 wherein the agonist is anadenosine A_(2A) receptor agonist.